Transcription Factor A, Mitochondrial Research Articles

Alterations of DNA methylation profile in peripheral blood of children with simple obesity

PurposeTo investigate the association between DNA methylation and childhood simple obesity.MethodsGenome-wide analysis of DNA methylation was conducted on peripheral blood samples from 41 children with simple obesity and 31 normal controls to identify differentially methylated sites (DMS). Subsequently, gene functional analysis of differentially methylated genes (DMGs) was carried out. After screening the characteristic DMGs based on specific conditions, the methylated levels of these DMS were evaluated and verified by pyrosequencing. Receiver operating characteristic (ROC) curve analysis assessed the predictive efficacy of corresponding DMGs. Finally, Pearson correlation analysis revealed the correlation between specific DMS and clinical data.ResultsThe overall DNA methylation level in the obesity group was significantly lower than in normal. A total of 241 DMS were identified. Functional pathway analysis revealed that DMGs were primarily involved in lipid metabolism, carbohydrate metabolism, amino acid metabolism, human diseases, among other pathways. The characteristic DMS within the genes Transcription factor A mitochondrial (TFAM) and Piezo type mechanosensitive ion channel component 1(PIEZO1) were recognized as CpG-cg05831083 and CpG-cg14926485, respectively. Furthermore, the methylation level of CpG-cg05831083 significantly correlated with body mass index (BMI) and vitamin D.ConclusionsAbnormal DNA methylation is closely related to childhood simple obesity. The altered methylation of CpG-cg05831083 and CpG-cg14926485 could potentially serve as biomarkers for childhood simple obesity.

Mitochondrial DNA replication stress triggers a pro-inflammatory endosomal pathway of nucleoid disposal.

Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. As a DAMP, mtDNA not only contributes to anti-viral resistance, but also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress caused by depletion of the mtDNA-packaging protein, transcription factor A, mitochondrial (TFAM) or during herpes simplex virus-1 infection exhibit elongated mitochondria, enlargement of nucleoids (mtDNA-protein complexes) and activation of cGAS-STING innate immune signalling via mtDNA released into the cytoplasm. However, the relationship among aberrant mitochondria and nucleoid dynamics, mtDNA release and cGAS-STING activation remains unclear. Here we show that, under a variety of mtDNA replication stress conditions and during herpes simplex virus-1 infection, enlarged nucleoids that remain bound to TFAM exit mitochondria. Enlarged nucleoids arise from mtDNA experiencing replication stress, which causes nucleoid clustering via a block in mitochondrial fission at a stage when endoplasmic reticulum actin polymerization would normally commence, defining a fission checkpoint that ensures mtDNA has completed replication and is competent for segregation into daughter mitochondria. Chronic engagement of this checkpoint results in enlarged nucleoids trafficking into early and then late endosomes for disposal. Endosomal rupture during transit through this endosomal pathway ultimately causes mtDNA-mediated cGAS-STING activation. Thus, we propose that replication-incompetent nucleoids are selectively eliminated by an adaptive mitochondria-endosomal quality control pathway that is prone to innate immune system activation, which might represent a therapeutic target to prevent mtDNA-mediated inflammation during viral infection and other pathogenic states.

IN VITRO BISPHENOL A EFFECT ON TFAM AND SIRT1 GENE EXPRESSION IN PORCINE OOCYTE MITOCHONDRIA

"Mitochondria are the main cellular organelle responsible for energy production, having an essential role in maintaining cellular homeostasis. In this study, the gene expression of TFAM (Transcription Factor A Mitochondrial) and SIRT1 (Silent Information Regulator) in sow oocytes cultured in vitro was assessed using the RT-qPCR reaction. The primers were tailored after our own design. The groups were: V1 (matured oocytes, control group), V2 (matured oocytes with hormones), V3 (medium supplemented with Bisphenol A), V4 (medium supplemented with Bisphenol A and hormones). Our findings reveal a reduction in SIRT1 activity following maturation in all experimental groups, while TFAM activity displayed more elevated levels, seemingly independent of the SIRT1 gene expression. The highest values of gene expression for TFAM and SIRT1 were obtained in V2 (supplemented with FSH and LH, - 0.277 and 0.010) and V4 (FSH, LH and bisphenol A - 0.272 and 0.015) without significant differences (p=0.941). Bisphenol A alone generated low values, presumably due to its endocrine disruptor action. We concluded that FSH/LH addition might rescue some of the TFAM expression during bisphenol treatment, but the mechanism might be independent of SIRT1."

FGF21 and autophagy coordinately counteract kidney disease progression during aging and obesity

ABSTRACT Chronic kidney disease (CKD) has reached epidemic proportions worldwide, partly due to the increasing population of elderly and obesity. Macroautophagy/autophagy counteracts CKD progression, whereas autophagy is stagnated owing to lysosomal overburden during aging and obesity, which promotes CKD progression. Therefore, for preventing CKD progression during aging and obesity, it is important to elucidate the compensation mechanisms of autophagy stagnation. We recently showed that FGF21 (fibroblast growth factor 21), which is a prolongevity and metabolic hormone, is induced by autophagy deficiency in kidney proximal tubular epithelial cells (PTECs); however, its pathophysiological role remains uncertain. Here, we investigated the interplay between FGF21 and autophagy and the direct contribution of endogenous FGF21 in the kidney during aging and obesity using PTEC-specific fgf21- and/or atg5-deficient mice at 24 months (aged) or under high-fat diet (obese) conditions. PTEC-specific FGF21 deficiency in young mice increased autophagic flux due to increased demand of autophagy, whereas fgf21-deficient aged or obese mice exacerbated autophagy stagnation due to severer lysosomal overburden caused by aberrant autophagy. FGF21 was robustly induced by autophagy deficiency, and aged or obese PTEC-specific fgf21- and atg5-double deficient mice deteriorated renal histology compared with atg5-deficient mice. Mitochondrial function was severely disturbed concomitant with exacerbated oxidative stress and downregulated TFAM (transcription factor A, mitochondrial) in double-deficient mice. These results indicate that FGF21 is robustly induced by autophagy disturbance and protects against CKD progression during aging and obesity by alleviating autophagy stagnation and maintaining mitochondrial homeostasis, which will pave the way to a novel treatment for CKD.

Melatonin attenuates sepsis-induced acute kidney injury by promoting mitophagy through SIRT3-mediated TFAM deacetylation

ABSTRACT A growing number of studies have shown that melatonin is a potent agonist of SIRT3 (sirtuin 3) and effectively protects mitochondria, suggesting melatonin as a promising therapeutic drug to treat sepsis-induced acute kidney injury (SAKI). In this study, we first included 48 sepsis patients for cross-sectional analysis and the result revealed that plasma melatonin levels are inversely proportional to the mitochondrial injury of renal tubular epithelial cells and directly proportional to the acute kidney injury recovery in sepsis patients. We further explored the effects of exogenous melatonin on SAKI and its mechanism in animal model of cecal ligation and puncture-treated mice, and cellular model of LPS-challenged HK-2 cells. The results demonstrate that the protective effect of melatonin on the progression of SAKI depends on SIRT3 activation as well as mitophagic flux. Mechanistically, TFAM (transcription factor A, mitochondrial)-K154 site deacetylation via SIRT3 is indispensable for melatonin-enhanced mitophagic flux on SAKI. Together, our study unveils a previously unappreciated mechanism of melatonin as agonists of SIRT3 to hold promise for treatment of patients with SAKI. Abbreviations AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E: hematoxylin and eosin staining; LCN2/NGAL: lipocalin 2; LPS: lipopolysaccharide; LTL: lotus tetragonolobus lectin; mKeima: mitochondria-targeted Keima; mtDNA: mitochondrial DNA; PAS: periodic acid – Schiff staining; RTECs: renal tubular epithelial cells; SAKI: sepsis-induced acute kidney injury; Scr: serum creatinine; SIRT3: sirtuin 3; TFAM: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine.

TFAM in mtDNA Homeostasis: Open Questions.

Transcription factor A, mitochondrial (TFAM) is a key player in mitochondrial DNA (mtDNA) transcription and replication [...]

Albizia julibrissin Exerts Anti-Obesity Effects by Inducing the Browning of 3T3L1 White Adipocytes.

This study investigated the effects of the Albizia julibrissin Leaf extracts (AJLE) on adipocytes using 3T3-L1 cells. AJLE inhibited adipogenesis by reducing the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteins (C/EBPs) that regulate enzymes involved in fat synthesis and storage, and subsequently reduced intracellular lipid droplets, glycerol-3-phosphate dehydrogenase (GPDH), and triglyceride (TG). AJLE also increased the expression of brown adipocyte markers, such as uncoupling protein-1 (UCP-1), PR/SET domain 16 (PRDM16), and bone morphogenetic protein 7 (BMP7) by inducing the differentiation of brown adipocytes, as shown by a decrease in the lipid droplet sizes and increasing mitochondrial mass. AJLE increased the expression of transcription factor A, mitochondrial (TFAM), mitochondrial DNA (mtDNA) copy number, and UCP-1 protein expression, all of which are key factors in regulating mitochondrial biogenesis. AJLE-induced browning was shown to be regulated by the coordination of AMPK, p38, and SIRT1 signaling pathways. The ability of AJLE to inhibit adipogenesis and induce brown adipocyte differentiation may help treat obesity and related diseases.

35 Years of TFAM Research: Old Protein, New Puzzles.

Transcription Factor A Mitochondrial (TFAM), through its contributions to mtDNA maintenance and expression, is essential for cellular bioenergetics and, therefore, for the very survival of cells. Thirty-five years of research on TFAM structure and function generated a considerable body of experimental evidence, some of which remains to be fully reconciled. Recent advancements allowed an unprecedented glimpse into the structure of TFAM complexed with promoter DNA and TFAM within the open promoter complexes. These novel insights, however, raise new questions about the function of this remarkable protein. In our review, we compile the available literature on TFAM structure and function and provide some critical analysis of the available data.

Rosiglitazone improves alveolar macrophage mitochondrial derangements induced by chronic alcohol consumption and infection with Klebsiella pneumoniae.

Abstract Objective: People with alcohol use disorders (AUDs) are 2–4 times more likely to develop lung infections, sepsis, and acute respiratory distress syndrome. Chronic alcohol misuse blunts alveolar macrophage (AM) immunity, in part, due to decreased peroxisome proliferator-activated receptor (PPAR)γ which increases cellular oxidative stress. Mitochondrial (MT) oxidative stress may impair AM phagocytosis; thus, we posited that chronic alcohol plus acute Klebsiella pneumoniae(Kp) dysregulates MT and treatment with rosiglitazone (RSG), a PPARγ ligand, reverses these effects to improve AM phagocytosis of Kp. Methods: AMs were isolated from 8–10-month-old male C57BL/6J mice fed ± ethanol (EtOH, 20% wt/vol in drinking water) for 12 wks ± intratracheal 2 × 10 4CFU of Kpin saline vehicle for the last 24 h of water or EtOH ± RSG (10 mg/kg/d in methylcellulose vehicle) via oral gavage 4 h prior to sacrifice (n=5/group). MT O 2•−and H 2O 2were assessed by fluorescence microscopy. mRNA and protein levels of PPARγ and MT dynamics mediators, transcription factor A mitochondrial (TFAM), dynamin-related protein 1 (DRP1), and mitofusin-2 (MFN2) were measured by qRT-PCR and cytoimmunostaining. Kpclearance by AMs was determined by plating lysed AMs on MacConkey agar. Results: EtOH feeding to mice increased MT oxidative stress; decreased PPARγ, TFAM, and MFN2 levels; and increased DRP1 levels in AMs. Kpexacerbated EtOH’s MT derangements by ~51–63% and impaired Kpclearance in AMs by 57.3 ± 11.2%. RSG treatment in vivoreversed EtOH- and Kp-induced MT derangements in AMs and restored AM clearance of Kp. Conclusion: RSG may provide a novel therapeutic strategy for mitigating AM MT derangements and immune dysfunction in AUD individuals infected with Kp. NIAAA R01-AA026086

Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis

Germinal center (GC) B cells undergo proliferation at very high rates in a hypoxic microenvironment but the cellular processes driving this are incompletely understood. Here we show that the mitochondria of GC B cells are highly dynamic, with significantly upregulated transcription and translation rates associated with the activity of transcription factor A, mitochondrial (TFAM). TFAM, while also necessary for normal B cell development, is required for entry of activated GC precursor B cells into the germinal center reaction; deletion of Tfam significantly impairs GC formation, function and output. Loss of TFAM in B cells compromises the actin cytoskeleton and impairs cellular motility of GC B cells in response to chemokine signaling, leading to their spatial disorganization. We show that B cell lymphoma substantially increases mitochondrial translation and that deletion of Tfam in B cells is protective against the development of lymphoma in a c-Myc transgenic mouse model. Finally, we show that pharmacological inhibition of mitochondrial transcription and translation inhibits growth of GC-derived human lymphoma cells and induces similar defects in the actin cytoskeleton.

Human cytomegalovirus infection activates NLRP3 inflammasome by releasing mtDNA into the cytosol in human THP-1 cells.

Human cytomegalovirus (HCMV) infection of monocytes results in the production of inflammatory cytokine through inflammasome. However, the mechanism of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in HCMV infection remains unclear. In this study, HCMV infection promoted the increase of mitochondrial fusion and caused mitochondrial dysfunction in THP-1 cells, including excessive reactive oxygen species production and decreased mitochondrial membrane potential (Δψm). Meanwhile, the expression of mitochondrial DNA (mtDNA)-binding protein TFAM (transcription factor A, mitochondrial) was decreased and mtDNA content in the cytoplasm was increased. Knockdown of TFAM caused an increase in mtDNA copy number in the cytoplasm and resulted in elevated NLRP3 expression, active caspase-1, and mature IL-1β. After a 3 h treatment with MCC950, an NLRP3 inhibitor, the increase of cleaved caspase-1 and mature IL-1β were suppressed. Besides, overexpression of TFAM inhibited the expression of NLRP3, cleaved caspase-1, and mature IL-1β. In addition, knockdown of NLRP3 inhibited the IL-1β process after HCMV infection. mtDNA-deficient cells showed a limited ability to produce NLRP3 and process IL-1β after HCMV infection. In conclusion, HCMV infection of THP-1 cells resulted in decreased mitochondrial TFAM protein expression and increased mtDNA release into the cytoplasm, which eventually led to the activation of NLRP3 inflammasome.

Adenosine Improves Mitochondrial Function and Biogenesis in Friedreich's Ataxia Fibroblasts Following L-Buthionine Sulfoximine-Induced Oxidative Stress.

Adenosine is a nucleoside that is widely distributed in the central nervous system and acts as a central excitatory and inhibitory neurotransmitter in the brain. The protective role of adenosine in different pathological conditions and neurodegenerative diseases is mainly mediated by adenosine receptors. However, its potential role in mitigating the deleterious effects of oxidative stress in Friedreich's ataxia (FRDA) remains poorly understood. We aimed to investigate the protective effects of adenosine against mitochondrial dysfunction and impaired mitochondrial biogenesis in L-buthionine sulfoximine (BSO)-induced oxidative stress in dermal fibroblasts derived from an FRDA patient. The FRDA fibroblasts were pre-treated with adenosine for 2 h, followed by 12.50 mM BSO to induce oxidative stress. Cells in medium without any treatments or pre-treated with 5 µM idebenone served as the negative and positive controls, respectively. Cell viability, mitochondrial membrane potential (MMP), aconitase activity, adenosine triphosphate (ATP) level, mitochondrial biogenesis, and associated gene expressions were assessed. We observed disruption of mitochondrial function and biogenesis and alteration in gene expression patterns in BSO-treated FRDA fibroblasts. Pre-treatment with adenosine ranging from 0-600 µM restored MMP, promoted ATP production and mitochondrial biogenesis, and modulated the expression of key metabolic genes, namely nuclear respiratory factor 1 (NRF1), transcription factor A, mitochondrial (TFAM), and NFE2-like bZIP transcription factor 2 (NFE2L2). Our study demonstrated that adenosine targeted mitochondrial defects in FRDA, contributing to improved mitochondrial function and biogenesis, leading to cellular iron homeostasis. Therefore, we suggest a possible therapeutic role for adenosine in FRDA.

β-Aminoisobutyric acid (L-BAIBA) is a novel regulator of mitochondrial biogenesis and respiratory function in human podocytes

Podocytes constitute an external layer of the glomerular filtration barrier, injury to which is a hallmark of renal disease. Mitochondrial dysfunction often accompanies podocyte damage and is associated with an increase in oxidative stress and apoptosis. β-Aminoisobutyric acid (BAIBA) belongs to natural β-amino acids and is known to exert anti-inflammatory and antioxidant effects. BAIBA has been reported to be involved in regulating mitochondrial dynamics, but unknown is whether BAIBA influences podocyte bioenergetics. The present study showed that human podocytes express the BAIBA receptor, Mas-related G protein-coupled receptor type D (MRGPRD), which is sensitive to BAIBA stimulation. The treatment of podocytes with L-BAIBA significantly increased their respiratory parameters, such as basal and maximal respiration, adenosine triphosphate (ATP) production, and spare respiratory capacity. We also found that L-BAIBA altered mitochondrial quantity, size, and shape, promoting organelle elongation and branching. L-BAIBA significantly upregulated peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) and transcription factor A mitochondrial (TFAM), indicating an increase in mitochondrial biogenesis. Our results demonstrate a novel regulatory mechanism of mitochondrial dynamics in podocytes, which may be important for maintaining their functions in the renal filtration barrier and prompting further investigations of preventing or ameliorating mitochondrial damage in podocytes in pathological states.

Quantitative assessment of mitophagy in irradiated cancer cells.

Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.

Role of Glycogen Mobilization and Mitochondrial Bioenergetics in Platelet Function, Hemostasis, and Thrombosis

Platelets are one of the most metabolically active cells in the bloodstream. They show metabolic flexibility, switching between glycolysis and oxidative phosphorylation (OxPhos) depending on oxygen tension and the availability of substrates. Platelets also have metabolizable glycogen granules that contribute to the energy pool upon activation. Our goal was to investigate platelet energy metabolism under normal physiological conditions to better understand the relative roles of the major ATP-generating processes (glycolysis, OxPhos, and glycogenolysis). To address the role of glycogen, we used 2 glycogen phosphorylase inhibitors and tested their effects on standard platelet functions e.g., secretion, aggregation, clot contraction, and thrombus formation under shear. We also measured the glycogen levels in resting and activated platelets and the presence of inhibitors. Consistent with previous studies, glycogen was utilized when platelets are activated with a ~25% decrease upon thrombin-mediated activation. Both inhibitors blocked the use of glycogen and led to a 3-fold increase in glycogen compared to controls. These data suggest the active turnover of glycogen in platelets. Our inhibitor studies indicated that glycogen is dispensable for low-energy processes like aggregation but contributed to secretion, clot contraction, and thrombus formation under shear. The inhibition was rescued by either increasing external glucose or bypassing glycolysis with added pyruvate. This study shows that the glycogen granules are metabolically active and contribute to the energy pool upon platelet activation; this pool is important for high-energy platelet functions like clot contraction. Deletion of platelet glucose transporters (GLUT1 and GLUT3) revealed an essential role of glucose metabolism in hemostasis in vivo. Glucose can power platelets via glycolysis alone or in combination with OxPhos (in the mitochondria). We sought to probe the relative importance of platelet mitochondrial bioenergetics in hemostasis and thrombosis. Previously used mitochondrial inhibitors (antimycin, oligomycin) are toxic and cannot be used for in vivo studies, thus we developed two novel mouse models with altered mitochondrial function using a platelet-specific deletion of TFAM and QPC. TFAM (Transcription Factor A Mitochondrial), is essential for the maintenance, transcription, and translation of mitochondrial DNA. Its deletion is expected to disrupt platelet mitochondrial DNA, which encodes 13 subunits of OxPhos. QPC is a subunit of ubiquinol-cytochrome c reductase complex III. Its deletion is expected to disrupt Complex III, which is part of the platelet mitochondrial respiratory chain. We confirmed TFAM deletion using Western blot analysis. Using the Seahorse Analyzer, we confirmed that deleting TFAM disrupted platelet mitochondrial bioenergetics in both resting and thrombin-stimulated platelets as seen as a ~40% decrease in Oxygen Consumption Rate (OCR). We also observed a significant decrease in ATP production indicating the contribution of OxPhos to ATP generation. Aggregation was not affected but in vitro clot contraction and thrombus formation under shear were defective (~58% increase in lag time, ~30% increase in AUC, and ~60% decrease in surface area coverage). Both the KO animals showed an increased tail-bleeding time, an increased occlusion time in the FeCl3 carotid injury model, and delayed hemostasis in a jugular puncture injury model with significantly higher rebleeding indicating that mitochondrial bioenergetics is important for clot stability. Using two novel mouse models with dysfunctional mitochondrial bioenergetics, we show that OxPhos is dispensable for low-energy demanding platelet functions such as aggregation but is important for secretion, clot contraction, hemostasis, and thrombosis (Table 1). Platelets show considerable plasticity in energy metabolism using both glycolysis and oxidative phosphorylation. Using mouse models and inhibitors, we show the relative importance of these energy-producing processes and the fuels and metabolic pathway choices made by platelets at different functional stages. Improving our understanding of platelet metabolism and the relative contributions of each pathway in the future could lead to a better understanding of how metabolic disorders can affect thrombotic risk in diabetic and obese patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondria dysfunction impairs Tribolium castaneum wing development during metamorphosis

The disproportionate growth of insect appendages such as facultative growth of wings and exaggeration of beetle horns are examples of phenotypic plasticity. Insect metamorphosis is the critical stage for development of pupal and adult structures and degeneration of the larval cells. How the disproportionate growth of external appendages is regulated during tissue remodeling remains unanswered. Tribolium castaneum is used as a model to study the function of mitochondria in metamorphosis. Mitochondrial dysfunction is achieved by the knockdown of key mitochondrial regulators. Here we show that mitochondrial function is not required for metamorphosis except that severe mitochondrial dysfunction blocks ecdysis. Surprisingly, various abnormal wing growth, including short and wingless phenotypes, are induced after knocking down mitochondrial regulators. Mitochondrial activity is regulated by IIS (insulin/insulin-like growth factor signaling)/FOXO (forkhead box, sub-group O) pathway through TFAM (transcription factor A, mitochondrial). RNA sequencing and differential gene expression analysis show that wing-patterning and insect hormone response genes are downregulated, while programmed cell death and immune response genes are upregulated in insect wing discs with mitochondrial dysfunction. These studies reveal that mitochondria play critical roles in regulating insect wing growth by targeting wing development during metamorphosis, thus showing a novel molecular mechanism underlying developmental plasticity.

NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α

Neuregulin-1 (NRG1)/erythroblastic leukaemia viral oncogene homologues 2 (ErbB2) pathway had been implicated in promoting differentiation and suppressing apoptosis of neuronal stem cells (NSCs) isolated from cochlear nucleus. In the current study, we aimed at determining the effects of NRG1/ErbB2 on mitochondrial (mt) function of NSCs. As expected, NRG1 increased the expression of mitofusin (Mfn) 1 and Mfn2 and decreased the expression of mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). However, after ErbB2 knockout, Mfn1 and Mfn2 expression decreased while Fis1 and Drp1 increased. Moreover, the increased mtDNA copy number and intracellular ATP level, elevated ATPase activities as well as decreased lactate production induced by NRG1 were partially reversed by ErbB2 knockout. Additionally, NRG1 treatment increased the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and upregulated the protein expression of catalase, manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM), which were also reversed by ErbB2 knockout. Furthermore, PGC-1α overexpression partially reversed the above effects of ErbB2 knockout. In conclusion, these findings suggest that the promotion of mitochondrial function of NRG1/ErbB2 axis is at least in part mediated by PGC-1α in NSCs from cochlear nucleus.

Herpesvirus-Mimicking DNAzyme-Loaded Nanoparticles as a Mitochondrial DNA Stress Inducer to Activate Innate Immunity for Tumor Therapy.

Virus-based immunotherapy is a promising approach to treat tumor. Closely mimicking the structure and sequential infection processes of natural viruses is highly desirable for effective tumor immunotherapy but remains challenging. Here, inspired by the robust innate immunity induced by herpesvirus, a herpesvirus-mimicking nanoparticle (named Vir-ZM@TD) is engineered for tumor therapy by mimicking the structure and infection processes of herpesvirus. In this biomimetic system, DNAzyme-loaded manganese-doped zeolitic imidazolate framework-90 (ZIF-90) nanoparticles (ZM@TD) mimic the virus nucleocapsid containing the genome; the erythrocyte membrane mimics the viral envelope; and two functional peptides, RGD and HA2 peptides, resemble the surface glycoprotein spikes of herpesvirus. Vir-ZM@TD can both effectively evade rapid clearance in the blood circulation and closely mimic the serial infection processes of herpesvirus, including specific tumor targeting, membrane fusion-mediated endosomal escape, and TFAM (transcription factor A, mitochondrial) deficiency-triggered mitochondrial DNA stress, as well as the release of manganese ions (Mn2+ ) from organelles into the cytosol, ultimately effectively priming cGAS-STING pathway-mediated innate immunity with 68% complete regression of primary tumors and extending by 32 days the median survival time of 4T1-tumor-bearing mice.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Abstract 2375: TFAM drives metabolic plasticity and cisplatin resistance in bladder cancer

Abstract Background: First-line therapy for locally advanced and metastatic bladder cancer (BC) consists of cisplatin-based chemotherapy, but its efficacy is limited by the emergence of resistance. We previously showed that resistance can emerge rapidly, in a mutation-independent manner, through epigenetic and transcriptional reprogramming associated with a metabolic shift towards oxidative phosphorylation (OxPhos). Based on these findings, we hypothesized that genes associated with mitochondrial activation may drive the transition to chemoresistance. Methods: Using our previously published (PMID: 21518726, 23784558, 32017074) model of bladder cancer plasticity, we FACS-sorted BC cell lines into highly aggressive, stem-like, drug-resistant side population (SP) and non-stem-like drug-sensitive non-side population (NSP). SP and NSP cells were analyzed by RNA-seq, and differentially expressed genes were entered into the Gene Transcription Regulation Database (GTRD; http://gtrd.biouml.org/) to identify candidate upstream regulators associated with OxPhos. For functional analysis, T24 and J82 BC cells were treated with cisplatin, with or without siRNA knockdown of candidate driver genes, followed by qRT-PCR, Western blot, FACS, and cell counts. Results: Differentially expressed genes between SP and NSP were analyzed by GTRD, which predicted Transcription Factor A, Mitochondrial (TFAM) as a key upstream driver of the phenotypic transition between the two cell states. Overexpression of TFAM in SP cells was confirmed by qRT-PCR (1.8-fold, p value <0.001). Cisplatin treatment (10μM) induced higher expression of TFAM mRNA and protein (1.6-fold, p value <0.001 and 1.7-fold, p value <0.001, respectively, at 72hr). Conversely, siRNA-mediated TFAM knock-down at 48hr reduced the number of SP cells (0.5-fold, p value 0.03), depleted mitochondrial DNA (0.7-fold, p value <0.001), and synergized with cisplatin to kill BC cells (coefficient of drug interaction: 0.57, p value <0.001). TCGA analysis revealed significantly higher TFAM expression in primary BC tumors vs. normal (p value <0.001) and in higher histological grade (p value 0.006). Conclusion: We demonstrate that drug-resistant BC cells overexpress TFAM, a known mitochondrial activator, and that more aggressive BC in TCGA is also marked by TFAM elevation. Importantly, cisplatin exposure induces TFAM expression which in turn drives a rapid shift towards a drug-resistant phenotype. Hence, TFAM may play a key role in non-mutation-mediated metabolic plasticity, permitting a subset of BC cells to persist in the face of cisplatin exposure. Further analysis of TFAM and its partners may elucidate new therapeutic targets to delay or avert drug resistance in BC. Citation Format: Maheen Iqbal, Tong Xu, Yi-Tsung Lu, Amir Goldkorn. TFAM drives metabolic plasticity and cisplatin resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2375.